Protein structural dynamics by single-molecule fluorescence polarization

Forkey, Joseph N.; Quinlan, Margot E.; Goldman, Yale E.

doi:10.1016/s0079-6107(00)00015-8

Cited by 122 publications

(81 citation statements)

References 93 publications

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“…A number of parameters that characterize single molecule emission are now routinely accessible at ambient temperatures, including emission intensity and polarization [12–13], fluorescence lifetime [14–16], and the emission spectrum [17–19]. Access to these parameters yields unique insights into distinct properties of single molecules, and enables the determination of the distributions of the relevant experimental parameters, revealing, for example, distinct sub-states and energetic levels in a heterogeneous population [20–21].…”

Section: Introductionmentioning

confidence: 99%

Room temperature excitation spectroscopy of single quantum dots

Blum

Schleifenbaum²,

Stopel³

et al. 2011

Beilstein J. Nanotechnol.

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SummaryWe report a single molecule detection scheme to investigate excitation spectra of single emitters at room temperature. We demonstrate the potential of single emitter photoluminescence excitation spectroscopy by recording excitation spectra of single CdSe nanocrystals over a wide spectral range of 100 nm. The spectra exhibit emission intermittency, characteristic of single emitters. We observe large variations in the spectra close to the band edge, which represent the individual heterogeneity of the observed quantum dots. We also find specific excitation wavelengths for which the single quantum dots analyzed show an increased propensity for a transition to a long-lived dark state. We expect that the additional capability of recording excitation spectra at room temperature from single emitters will enable insights into the photophysics of emitters that so far have remained inaccessible.

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Section: Introductionmentioning

confidence: 99%

Room temperature excitation spectroscopy of single quantum dots

Blum

Schleifenbaum²,

Stopel³

et al. 2011

Beilstein J. Nanotechnol.

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show abstract

“…For single-molecule analysis, Goldman and coworkers have altered their fluorescence excitation scheme to use prism-type total internal reflection (TIR) illumination (Forkey et al , 2000, 2005, 2003). TIR excitation has the benefit that only a thin slice of the sample, up to about 100 nm, from a glass–water interface is illuminated, reducing background fluorescence.…”

Section: Methodsmentioning

confidence: 99%

Structure and Dynamics of the Kinesin–Microtubule Interaction Revealed by Fluorescence Polarization Microscopy

Sosa

Asenjo

Peterman

2010

Methods in Cell Biology

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Fluorescence polarization microscopy (FPM) is the analysis of the polarization of light in a fluorescent microscope in order to determine the angular orientation and rotational mobility of fluorescent molecules. Key advantages of FPM, relative to other structural analysis techniques, are that it allows the detection of conformational changes of fluorescently labeled macromolecules in real time in physiological conditions and at the single-molecule level. In this chapter we describe in detail the FPM experimental set-up and analysis methods we have used to investigate structural intermediates of the motor protein kinesin-1 associated with its walking mechanism along microtubules. We also briefly describe additional FPM methods that have been used to investigate other macromolecular complexes.

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“…The motions can be quantified by calculating analytical expressions for the requisite polarized fluorescence intensities that take into account the expected correlations between excitation and emission, and other characteristics of the instrument and optics [14, 19, 47, 100, 101]. …”

Section: Detecting Timescales Of Motionmentioning

confidence: 99%

Single molecule optical measurements of orientation and rotations of biological macromolecules

Shroder

Lippert

Goldman

2016

Methods Appl. Fluoresc.

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The subdomains of macromolecules often undergo large orientation changes during their catalytic cycles that are essential for their activity. Tracking these rearrangements in real time opens a powerful window into the link between protein structure and functional output. Site-specific labeling of individual molecules with polarized optical probes and measuring their spatial orientation can give insight into the crucial conformational changes, dynamics, and fluctuations of macromolecules. Here we describe the range of single molecule optical technologies that can extract orientation information from these probes, we review the relevant types of probes and labeling techniques, and we highlight the advantages and disadvantages of these technologies for addressing specific inquiries.

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Protein structural dynamics by single-molecule fluorescence polarization

Cited by 122 publications

References 93 publications

Room temperature excitation spectroscopy of single quantum dots

Room temperature excitation spectroscopy of single quantum dots

Structure and Dynamics of the Kinesin–Microtubule Interaction Revealed by Fluorescence Polarization Microscopy

Single molecule optical measurements of orientation and rotations of biological macromolecules

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